Method for controlling O-desulfation of heparin and compositions produced thereby

ABSTRACT

Methods of making and using, as prophylactics or therapeutics for preventing or treating certain diseases including cancer, O-desulfated heparin compositions, preferably 2-O, 3-O desulfated heparin compositions, wherein the methods permit regulating the degree of 2-O, 3-O desulfation to produce compositions that are variably desulfated upto about and 75% desulfated at the 2-O and 3-O positions, respectively.

This patent application is a continuation of U.S. patent applicationSer. No. 08/300,291, filed Sep. 1, 1994 now abandoned, which is acontinuation-in-part of Ser. No. 08/210,847, filed Mar. 21, 1994 nowabandoned. Ser. No. 08/210,847 is a continuation-in-part of U.S. Ser.No. 08/994,804 filed Dec. 22, 1992, abn., U.S. Pat. No. 5,296,471,issued on Mar. 22, 1994. Priority is hereby claimed per 35 U.S.C. §120.

FIELD OF THE INVENTION

This invention relates to methods of making O-desulfated heparincompositions, preferably 2-O, 3-O desulfated heparin compositions,wherein the methods permit regulating the degree of 2-O, 3-O desulfationto produce compositions that can be variably desulfated including about99% and 75% desulfated at the 2-O and 3-O positions, respectively. Suchcompositions, have significant anti-cancer activity in vivo,substantially lack anti-coagulant activity, inhibit plateletaggregation, exhibit reduced binding to bFGF, and have anti-angiogenicand heparanase inhibitory activity. The compositions are useful fortreating various diseases, including cancer.

Abbreviations

The following abbreviations are used for monosaccharides or formonosaccharides residues included in oligomers: D-glucuronic acid=GlcA;L-iduronic acid=IdoA; D-glucosamine=GlcNH₂ ;N-acetyl-D-glucosamine=GlcNAc; D-glucosamine N-sulfate=GlcNS;2,5-anhydromannose=Aman; 2,5-anhydromannitol=AManHh.

Abbreviations that are used to denote disaccharide residues obtained inthe analysis of heparin compositions described herein are as follows:ISMS is defined as IdoA (2-sulfate)→AManH (6-sulfate); GMS₂ is definedas GlcA→AManH (3,6-disulfate); IS is defined as IdoA (2-sulfate)→AManH(6-sulfate)+IdoA (2-sulfate)→AManH.

In designating each saccharide residues below the appropriateabbreviation, the location of the O-linked sulfate residues is indicatedby "S" and the number of the position of sulfation where the sulfateresidue is linked to oxygen on the sugar residue. In the designationsfor heparin structure, also, the positions involved in the alpha andbeta anomeric linkages are as those conventionally found in heparin, α(glucosamine→uronic) and β (uronic→glucosamine), and the D or Lconfigurations as conventionally found pertains. The locations of thesulfates are shown below the abbreviation for the sugar to which theyapply, thus, for example, ##STR1## refers to a disaccharide composed ofL-iduronic acid and D-glucosamine N-sulfate-linked β(1-4) with sulfatesconnected respectively at the 2 and 6 positions of the sugar residues.

Background

Heparin

Heparin/heparan sulfate is a member of a class of polysaccharides knownas glycosaminoglycans (GAG). These materials are copolymers ofalternating hexosamine and aldouronic acid residues which are found insulfated forms and are synthesized as proteoglycans. In the compositionsof interest herein, heparan sulfate and heparin, the hexosamine whichpredominates is N-acetylated or N-sulfated glucosamine (GlcNAc andGlcNS). The aldouronic acid is mostly L-iduronic in heparin and mostlyD-glucuronic acid in heparan sulfate. Heparan sulfate is commonlyconsidered to have a higher proportion of glucuronic acid than heparin.

Problems of heterogeneity in preparations of heparan sulfate or heparinisolated from tissues make sharp distinctions difficult. Conventionalheparin (used as an anticoagulant) has a molecular weight of 5-25 kd andis extracted as a mixture of various chain lengths by conventionalprocedures. These procedures involve autolysis and extraction ofsuitable tissues, such as beef or porcine lung, intestine, or liver, andremoval of nonpolysaccharide components. The molecular weight of thechains in the extract is significantly lower than the 60-100 kd known toexist in the polysaccharide chains of the heparin proteoglycansynthesized in the tissue. The GAG moiety is synthesized bound to apeptide matrix at a serine or threonine residue through atetrasaccharide linkage region of the sequenceD-GlcA-D-Gal-D-Gal-D-Xyl→protein, which is then elongated at the D-GlcAresidue with alternate additions of GlcNAc and GlcA. The polymerundergoes epimerization at certain of the GlcA residues to give IdoA,and subsequent sulfation.

Due to their chemical similarity, isolated "heparin" may containconsiderable mounts of what might otherwise be classified as heparansulfate.

Modified Desulfated Heparins

Several investigators have described the preparation of desulfatedheparin. For instance, partially N-desulfated heparins are shown byVelluz, in C. R. Acad. Sci. Paris (1959) 247:1521, Sache, in ThrombosisRes. 55-247 (1989), and by Inoue in Carbohyd. Res. (1976) 46:87.

Other investigators have described the N-acetylation of N-desulfatedheparin. See, Purkenson, J. Clin. Invest. (1986) 81:69; Inoue, Carbohyd.Res. (1976) 46:87; and Ayotte, Carbohyd. Res. (1986) 145:267-277.

Further, Jaseja, M. et al., have reported that alkaline treatment ofheparin results in a sequence of specific transformations that involvethe loss of 2-O-sulfate. See, Can. J. Chem. (1989) 67:1449-1456. Theseinvestigators studied the alkaline desulfation of beef lung heparin. Theinstant invention presents methods for realizing defined compositions of2-O, 3-O desulfated heparin from a chemically less homogenous source ofheparin, hog mucosal heparin.

Three different transformations were characterized by Jaseja, M. et al.,above, depending on the reaction conditions. The first, lyophilizationof a mildly alkaline (pH 11.2-11.5) solution of beef lung heparin causedthe partial conversion of 2-O-sulfated IdoA residues to non-sulfateduronic acid residues possessing the α-L-gulo configuration, and a2,3-oxirane functionality (characterized by resonances in the ¹³ C-nmrspectrum at 53.5 and 54.0 ppm, and assigned to the C-2 and C-3 carbonsof the new uronic acid residue). The conversion to the oxirane underthese conditions is typically incomplete, with the highest reportedconversion of 65-70% of the IdoA2S in a beef lung heparin. The productsare stable and the oxirane could be isolated. The other 2-O-sulfatedIdoA residues are unaltered, or appear as non-sulfated IdoA.

The second transformation described by Jaseja, M. et al., above, occurswhen the oxirane is heated in alkaline solution. A product is producedthat has a reduced IdoA-2-O-sulfate content, and a new uronic acidconstituent appears which is isolatable. This second reaction wasfollowed by ¹³ C-nmr spectroscopy, and the disappearance of theresonances assigned to the oxirane carbons was accompanied by theappearance of new resonances distinct from IdoA. Resonances attributedto glucosamine residues were reportedly unaltered. This new residue waspostulated to be α-L-galacturonic acid. This was later confirmed bydetailed nmr analysis and chemical methods (Perlin, Carbohydr. Res.(1990) 200:437-447).

Rej and Perlin also reported (Carbohydr. Res. (1990) 200:437-447) thatIdoA2S residues in heparin could be directly converted to the L-galactoisomer by heating an aqueous solution of heparin containing 0.1M sodiumcarbonate at 100°-110° C. The extent of this direct reaction was notwell characterized, but typically yielded products with reducedanticoagulant activities (70 u/mg USP units) relative to the parent beeflung heparin (126 u/mg). The IdoA 2-O-sulfate residues were the onlyresidues reported to be involved in this reaction. U.S. Pat. No.5,104,860 claims similar products from reaction of alkaline heparinsolutions at elevated temperatures having reduced anticoagulant andantithrombotic activities, useful for treating nephrolithiases. Thesecompounds are characterized by the presence of a ¹³ C-nmr resonance at101.3 ppm, and optical rotation values between +15° and +40°, a reducedsulfate content (6-9% vs 10.6-11.6%), a sulfate:carboxylate ratio ofabout 1.2-1.7, and some free amine groups (0.4-2.1%). The relativecontent of native (IdoA2S) and transformed (L-GalA) residues was notprovided for any of the products claimed.

The third transformation described by Jaseja, M. et al., above, of theoxirane containing heparin derivative, occurs when an alkaline solution(pH 12.5-12.8) of the epoxide is lyophilized. The products of thistreatment were characterized by a reduced 2-O-sulfated IdoA content, anda corresponding increase in the amount of IdoA. It was also shown thattreatment of native heparin trader the same conditions yielded similarproducts, thus resulting in apparent direct 2-O desulfation. Theproducts obtained from this third reaction sequence had variable extentsof 2-O-desulfation. It is important to point out that one feature of theinvention described below is the description of methods that permitcontrolling the amount of 2-O desulfation.

It is noteworthy that Perlin later reported (Perlin, Carbohydr. Res.(1992) 228:29-36.) a study using a model heparin compound, methyl2-deoxy-2-sulfamino-a-D-glucopyranoside 3-O-sulfate, in which it wasalkaline treated similar to heparin. The work was conducted to determineif the 3-O-sulfate groups from glucosamine residues of heparin would belost during the alkaline lyophilization. The 3-O-sulfated compound wasrecovered unaltered from the reaction, leading to the conclusion thatthe 3-O-sulfate group in heparin is similarly unaffected.

It is further noteworthy that although Perlin describes heparincompositions that are 2-O desulfated, and methods to produce suchcompositions, he does not show a method to control the degree of 2-O,3-O desulfation, nor does he show or suggest methods for producingcompositions that can be variably desulfated including about 99% and75%, or greater, desulfated at the 2-O and 3-O positions, respectively.Indeed, the work of both Jaseja, M. et al., above, and Perlin, above,suggest that only 2-O desulfation of heparin occurs under their reactionconditions, and moreover, that the extent of 2-O desulfation is highlyvariable.

U.S. Pat. No. 5,010,063 claims an epoxy heparin prepared by heating analkaline solution of heparin. European patent application 483 733 claimsan "epoxy heparide" formed from reaction of an over N-acetylated heparinin a sodium hydroxide solution, containing hydrogen peroxide, atelevated temperatures.

Non-Anticoagulant Heparin

There is a body of art that describes the production ofnon-anticoagulant heparin. Most of the publications describenon-anticoagulant heparin produced from depolymerized heparin/heparansulfate, and separation of products by size. In a generally usedprocedure, the heparin starting material is depolymerized in thepresence of nitrous acid with or without pretreatment to remove N-acetylgroups from any GlcNAc residues present. Nitrous acid, under theappropriate conditions, cleaves at the linkage between a GlcNS or GlcNH₂residue and the uronic acid residue through which it is linked through aglucosamine α (1-4) uronic acid linkage. If the heparin has beendeacetylated, all of the glucosamine→uronic acid residues aresusceptible and complete depolymerization results in disaccharides. Ifthe heparin has not been deacetylated, the glucosamine→uronic acidresidues wherein the glucosamine is acetylated are resistant, and bothdisaccharides and tetrasaccharides and small amounts of higheroligosaccharides containing the resistant linkage result. In all cases,the glucosamine residue at the reducing terminus of the disaccharide ortetrasaccharide is convened to a 2,5-anhydromannose in the course ofcleavage. This residue may further be reduced to the corresponding2,5-anhydromannitol. These methods have been described by Bienkowski, M.J. and Conrad, H. E., J Biol Chem (1985) 260:356-365; Guo, Y. et at.,Anal Biochem (1988) 168:54-62; and Guo, Y. and Conrad, H. E., AnalBiochem (1989) 176:96-104. These latter methods are useful in analyzingthe structure of heparin and in assessing the results of varioustreatments of the heparin chains. Further, there have been considerableattempts to use the products of degradation of heparin from bothcomplete and partial digestion with nitrous acid as described in theforegoing papers, or from heparinase digestion or from periodateoxidation followed by β-elimination. All of these processes can generatelow molecular weight heparins for therapeutic use.

An example of non-anticoagulant depolymerized low molecular weightheparin is described in U.S. Pat. No. 4,990,502. It shows the treatmentof heparin with periodate, followed by depolymerization with base, andreduction of the aldehydes generated in the periodate treatment. Theresulting material is said to contain a mixture of polymers containing17-33 residues and containing a multiplicity of residues of the formula

    ______________________________________                                        IdoA-GlcAc   or            IdoA-GlcNS                                         2S                         2S                                                 ______________________________________                                    

wherein the glucosamine residue is sulfated at the 3 and/or 6 positionin an arbitrary manner, and wherein some of the IdoA residues may bereplaced by cleaved IdoA or GlcA residues resulting from the periodateoxidation. These shortened polymeric chains are said to lack the bindingsite for ATIII but to be capable of inhibiting smooth muscleproliferation and to have physiological activities that includeacceleration of tissue repair, prevention of atherogenous lesions, andprevention of the development of metastasis.

Treatment of heparin/heparan sulfate with periodate has also beenreported by others. For instance, Fransson, L. A. and Lewis, W., FEBSLett (1979) 97:119-123, describe a variety of conditions relating to thetreatment of heparin/heparan sulfate with periodate and reduction bysodium borohydride or fragmentation in alkaline medium. Further,Fransson, L. A. et al., Carbohydrate Res (1980) 80:131-145, studied thechemistry of various forms of heparin produced with periodate. In onestudy, the treatment with periodate is followed by β-elimination in baseto produce fragmentation. They further reported the treatment of heparinwith periodate followed by partial acid hydrolysis which results infragmentation of the chains and partial destruction of the functionalgroups.

Another example of a non-anticoagulant heparin is described by Casu, B.et al., Arzneim Forsch/Drug Res (1986) 36:637-642. They studied theeffect of periodate oxidation on the anti-lipemic (lipoproteinlipase-releasing) activity of heparin. In this study, the heparin wasoxidized with periodate and the products were reduced with borohydride.Although the authors stated that the product has the same molecularweight as the starting material, it is apparent from the figurespresented in the paper that there is significant depolymerization.

PCT/SE92/00243 shows a non-anticoagulant heparin that has a molecularweight larger than the heparin starting material, and that is producedby periodate oxidation, partial depolymerization by alkali, andsubsequent borohydride reduction.

Finally, the 2-O desulfated heparin compositions described by Jaseja, M.et al., in Can. J. Chem. (1989) 67:1449-1456, have non-anticoagulantactivity.

It is important to note, that although non-anticoagulant heparins areknown in the art, the art does not teach a method for producingnon-anticoagulant heparins that can be about 99% and 75% or greaterdesulfated at the 2-O Ido A and 3-O GlcN positions, respectively.

Biological Properties of Non-Anticoagulant Heparins

Aside from their non-anticoagulant activity, NAC heparins have certainother novel biological properties. Some of these are described below.

Inhibition of Heparanase

The metastatic spread of tumor cells throughout the body is thought tobe facilitated by enzymes secreted by tumor cells that degradecomponents of the basement membrane, thereby allowing tumor cells todisseminate via the circulation. One such enzyme isendo-β-D-glucuronidase, or heparanase, which degrades heparan sulfateglycosaminoglycans. Heparan sulfate is a prominent component ofparenchymal cell basement membranes.

PCT patent application, WO 92/01003, shows that certainnon-anticoagulant heparins act as heparanase inhibitors, and that theymay be effective in lessening or preventing lung colonization bymetastatic cell variants. The non-anticoagulant heparins were preparedfrom heparin by N-desulfation followed by N-acetylation, or N,Odesulfation followed by N-desulfation. Hence, the non-anticoagulantheparins described in the above PCT application are distinct from the2-O, 3-O desulfated heparin compositions of the instant invention.

Inhibition of Angiogenesis

Angiogenesis is the process whereby new blood vessels are produced. Itis a process that may be associated with certain diseases, includingarthritis, and the growth and metastasis of tumors. See, Mitchell andWilks, Annual Reports in Medicinal Chemistry (Academic Press 1992)27:139-148; Chapter 15.

Compounds that stimulate or inhibit angiogenesis can be identified usingseveral assays known in the art. Perhaps the easiest assay to use is thechicken chorioallantoic membrane (CAM) assay. With this assay it hasbeen shown that certain heparinoids inhibit angiogenesis whenadministered with certain angiostatic steroids. Folkman and Ingber, Ann.Surg. (1987) 206:374, Folkman et al., Science (1983) 221:719.

Inhibition of bFGF

Heparin or certain NAC heparins are known to bind bFGF with concomitantmodulation of bFGFs mitogenic activity. The bFGF binding properties ofcertain heparins or heparin like molecules are described in thispublication. For example, the results of a cell based competitivebinding assay showed that there is little inhibition of binding of bFGFto a target cell by chemically modified heparin including completelydesulfated, N-sulfated heparin, and N-desulfated, N-acetylated heparin.

Assays for measuring the effect of heparinoids on bFGF are known in theart. A cell based competitive binding assay is described by Ishihara, M.et al., Anal Biochem (1992) 202:310-315.

Platelet Inhibition

Heparin's best known property is its anti-coagulant activity, which isevidenced by the ability of heparin to prolong the bleeding time inanimals. This occurs because heparin binds to the protease pro-inhibitorantithrombin III via its specific antithrombin III binding region. This,in turn, ultimately blocks the blood clotting cascade. Heparin is alsoknown to have an anti-thrombotic effect, and at least in part this is aresult of heparin's capacity to inhibit platelet aggregation.Interference with platelet aggregation causes a significant bleedingliability in some patients. Certain NAC heparins exhibit bothnon-anticoagulant activity and inhibit platelet aggregation. See, forexample, co-owned U.S. Pat. No. 5,280,016, issued Jan. 18, 1994, or PCTPatent Application No. US92/02516, filed Mar. 27, 1992.

BRIEF DESCRIPTION OF THE INVENTION

In one aspect, the invention is directed to methods of O-desulfatingheparin, preferably to produce 2-O, 3-O desulfated heparin compositions.The methods permit controlling the degree of 2-O, 3-O desulfation suchthat compositions can be produced that have a desired amount ofdesulfation including upto about 99% or 75% or greater desulfated at the2-O and 3-O positions, respectively. The compositions have the followingunique properties; anti-cancer activity, substantially no anticoagulantactivity, inhibit platelet aggregation, reduced binding to bFGF relativeto heparin, and heparanase and angiogenic inhibitory activity.

A second aspect of the invention is directed to substantiallynon-fragmented heparin compositions that can be 99% or 75% or greaterdesulfated at the 2-O and 3-O positions, respectively.

A third aspect of the invention is directed to 2-O, 3-O desulfatedheparin fragments derived by chemical modification of heparin whereinthe fragments can be 99% or 75% or greater desulfated at the 2-O and 3-Opositions, respectively.

A fourth aspect of the invention is directed to methods of producingsubstantially unfermented 2-O, 3-O desulfated heparin compositions fromheparin, wherein the compositions can be 99% or 75% or greaterdesulfated at the 2-O and 3-O positions, respectively, via an alkalinemediated chemical reaction having a bivalent cation in the reactionmixture.

A fifth aspect of the invention is a description of methods of makingcompositions of 2-O, 3-O desulfated heparin fragments from heparin,wherein the fragments can be 99% or 75% or greater desulfated at the 2-Oand 3-O positions, respectively, via an alkaline mediated chemicalreaction.

A sixth aspect of the invention is directed to methods of preventing ortreating disease by administering to an animal host compositions ofsubstantially unfragmented 2-O, 3-O desulfated heparin, or 2-O, 3-Odesulfated heparin fragments wherein the compositions can be 99% or 75%or greater desulfated at the 2-O and 3-O positions, respectively.

A seventh aspect of the invention is the production of 2-O, 3-Odesulfated heparin, or 2-O, 3-O desulfated heparin fragments having freeamine groups that can be reacted with suitable reagents to yieldN-modified 2-O, 3-O desulfated heparin analogues.

These and other aspects of the invention will be more fully understoodupon a detailed consideration of the invention presented below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the effect of copper II on the alkaline desulfation ofheparin.

FIG. 2 shows the % APTT activity and % residual IS remaining of 2-Odesulfated heparin compositions of the instant invention as a functionof the ratio of copper to iduronic acid (2S) in the reaction mixture.The Cu (II) concentration is given in mmoles.

FIG. 3 compares the heparanase inhibitory activity of 2-O, 3-Odesulfated heparin produced by the methods of the instant invention,and, 2-O desulfated heparin produced by the methods of Jaseja, M., etal., Can. J. Chem. (1989) 67:1449-1456.

FIG. 4 shows the anti-tumor activity of 2-O, 3-O desulfated heparin innude mice on the highly metastatic pancreatic adenocarcinoma tumor cellline, CAPAN.

FIG. 5 shows the effect of sodium hydroxide concentration on 2-O, 3-Odesulfation of heparin fragments.

MODES OF CARRYING OUT THE INVENTION

In its most general form, the instant invention relates to compositionsand methods of producing the compositions, wherein the compositionsconsists of substantially unfragmented 2-O, 3-O desulfated heparin, or2-O, 3-O desulfated heparin fragments. The methods permit controllingthe per cent of 2-O, 3-O desulfation such that it can be about 99% or75%, or greater, at these positions, respectively.

Throughout the specification reference is made to certain scientificpublications, patents or patent applications. It is the intent of theapplicants that these references be incorporated in their entirety intothe application.

Understanding the invention will be facilitated by a brief discussion ofcertain of the technical terms used throughout the specification.

By "heparin/heparan sulfate" or "heparin" is meant a preparationobtained from tissues in a manner conventional for the preparation ofheparin as an anticoagulant or otherwise synthesized and correspondingto that obtained from tissue. See Conrad, H. E., Heparin and RelatedPolysaccharides, Vol. 56, p. 18 of Annals of N.Y., Academy of Sc., Jun.7, 1989, incorporated herein by reference. This preparation may includeresidues of D-glucuronic acid (GlcA), as characteristic of heparansulfate as well as iduronic acid (IdoA) as characteristic of heparin.However, even though both GlcA and IdoA are present in both, they arepresent in different proportional amounts. The IdoA/GlcA ratio rises asheparan sulfate becomes more heparin-like. As described in theBackground section above, the conversion of D-glucuronic acid toL-iduronic acid is a result of epimerization at the 5 carbon of GlcAresidues in a heparan-type intermediate. This sequence of steps involvedin such epimerization and conversion is understood in the art. To theextent that full conversion has not been made, heparan sulfatecharacteristics remain in the preparation. Because the precise nature ofthe polymeric chains in the preparations of heparin is not generallydetermined, and varies from preparation to preparation, the term"heparin/heparan sulfate" or "heparin" is intended to cover the range ofmixtures encountered. Perhaps the main feature which distinguishesheparan sulfate from heparin is that the latter has anti-coagulantactivity.

By heparin fragments, or low molecular weight heparin, is meant heparinthat has been treated with any one of a number of reagents and methodsthat depolymerize heparin with a average molecular weight of 5-30 kd tocompositions that have average molecular weights of 2-6.5 kd. Suchreagents and methods are known in the art, and examples would includenitrous acid depolymerization, benzylation followed by alkalinedepolymerization, peroxidative depolymerization, alkaline treatment, andenzymatic depolymerization with heparinase. See, Hirsh, J. and Levine,M., Blood (1992) 79:1-17.

The "heparin/heparan sulfate" or "heparin" preparation can be obtainedfrom a variety of mammalian tissues, including, if desired, humantissue. Generally, porcine or bovine sources are used, and vascularizedtissues are preferred. A preferred source of heparin starting materialis porcine intestinal mucosa, and preparations labeled "heparin"prepared from this tissue source are commercially available. In general,the heparin starling material is prepared from the selected tissuesource by allowing the tissue to undergo autolysis and extracting thetissue with alkali, followed by coagulation of the protein, and thenprecipitation of the heparin-protein complex from the supernatant byacidification. The complex is recovered by reprecipitation with a polarnonaqueous solvent, such as ethanol or acetone or their mixtures, andthe fats are removed by extraction with an organic solvent such asethanol and proteins by treatment with a proteolytic enzyme, such astrypsin. Suitable procedures for the preparation of the heparin startingmaterial are found, for example, in Charles, A. F., et al., Biochem J(1936) 30: 1927-1933, and modifications of this basic procedure are alsoknown, such as those disclosed by Coyne, E., in Chemistry and Biology ofHeparin (1981 ) Elsevier Publishers, North Holland, N.Y., Lunblad, R. L.et al., eds.

"NAC-heparin" refers to a mixture of substantially non-anticoagulant,non-fragmented heparin obtained by subjecting commercially availableheparin to one or more chemical treatments.

"Heparinoid" refers to glycosaminoglycans containing a 2-O-sulfatedresidue, including but not limited to heparin, chondroitin sulfates,dermatan sulfate, and NAC-heparin.

It is important to note that the disaccharide analysis of thecompositions described and claimed herein are those presented by Guo andConrad, Anal. Biochem. (1988) 168, 54-62. Such methods can detect 99% orgreater loss of sulfate from the 2 position of IdoA. However, because ofthe low level of 3-O-sulfated GlcN residues present in hog mucosaheparin the methods do not allow reliable detection of 3-O desulfationgreater than 75%.

In its general form the methods of the instant invention permitcontrolling the degree of 2-O, 3-O desulfation of unfragmented heparin,or heparin fragments, to yield compositions of substantiallyunfragmented 2-O, 3-O desulfated heparin, or 2-O, 3-O desulfated heparinfragments having a desired per cent of desulfation. The desulfationprocess can generally be performed using (a) methods involvinglyophilization to accomplish the reaction, or (b) methods involvingforming a paste of solid heparinoid with a base, preferably solid sodiumor potassium hydroxide.

(a) This method consists of dissolving commercially available heparin,preferably Ming Han heparin, 170U/mg, or heparin fragments, alsoreferred to as low molecular weight heparin (LMW heparin), in an aqueousalkaline solution (0.1-5.0% heparin or heparin fragments), in thepresence of metal ions capable of regulating the extent of desulfation.Such ions include bivalent metal ions. The preferred ions are calciumand copper. Monovalent ions would also perform satisfactorily but wouldbe used at higher concentrations than bivalent ions. Copper is the mostpreferred ion. Calcium and copper can be supplied in a variety of formsincluding CaCl₂, and CuSO₄. The ions are believed to interact with the2-O and 3-O sulfate groups in a manner not yet fully understood toeffectively shield them from desulfation. The degree of protection fromdesulfation is a function of ion concentration, which is elaborated morein the discussion below.

Hydroxide ion is present in the reaction; preferably in the form of anaqueous solution of an alkaline earth or alkali metal salt, to increasethe pH to a level that initiates 2-O, 3-O desulfation of either heparin,or heparin fragments. One skilled in the art, using well knownanalytical techniques, including nuclear magnetic resonance (NMR)spectroscopy, will know to choose the preferred pH, reaction time, andother experimental parameters, by monitoring the loss of 2-O sulfatefrom iduronic acid. Similarly, the loss of 3-O sulfate from GlcN can bemonitored as a function of reaction conditions, but as mentioned above,its loss cannot, however, be reliably quantitated greater than 75%. Forexample, the products can be identified using ¹ H- and ¹³ C-nmrspectroscopy with detailed compositional characterization performed byHPLC analysis of nitrous acid generated disaccharides. Guo and Conrad,Anal. Biochem. (1988) 168:54-62; see also, Jaseja, M. et al., Can. J.Chem. (1989) 67:1449-1456. The reaction mixture is frozen, lyophilizedto dryness, dissolved in water and excess hydroxide ion removed.

More specifically, heparin or heparin fragments, are dissolved in waterto make a 0.1-5.0% solution. A variety of commercially availableheparins may be used. The preferred heparin is Ming Han heparin, 170U/mg. Depending on the degree of 2-O, 3-O desulfation desired a knownamount of copper is added. A reducing agent (i.e. NaBH₄) may be added toprevent fragmentation of heparin. Indeed, because heparin isdepolymerized at elevated pHs, a reducing agent is preferably added tomaintain heparin in a substantially unfragmented form, preferably atpH's greater than 12.

Hydroxide ion is added to make the solution 0.05-1.0M with a preferredpH of 11-14. The preferred concentrations of hydroxide ion and heparinare 0.4M and 2%, respectively. Under these conditions, the 2-O, 3-Odesulfated compositions are less than 10% depolymerized when compared tothe heparin starting material. The solution is frozen and lyophilized todryness. The residue is dissolved in water and excess hydroxide ion isremoved, preferably using an ion-exchange resin (H+), or neutralizationwith acid. An aqueous solution of acetic or other mineral acids ispreferred. The pH is raised to between 8-9 with sodium bicarbonate toform the sodium salt. After exhaustive dialysis or ultrafiltration, thesolution is lyophilized to give substantially unfragmented 2-O, 3-Odesulfated heparin (less than 10% of the starting material isdepolymerized), or 2-O, 3-O desulfated heparin fragments. Alternately,the product can be precipitated from solution by the addition of ethanolusing procedures well known in the art.

(b) This method consists of mixing commercially available heparin,preferably Ming Han heparin, 170 U/mg, or heparin fragments, alsoreferred to as low molecular weight heparin (LMW heparin), with base,preferably solid sodium hydroxide (0.2-0.8 g NaOH/g of heparin orheparin fragments), in the presence of metal ions capable of regulatingthe extent of desulfation. Such ions include bivalent metal ions. Thepreferred ions are calcium and copper. Monovalent ions would alsoperform satisfactorily but would be used at higher concentrations thanbivalent ions. Copper is the most preferred ion. Calcium and copper canbe supplied in a variety of forms including CaCl₂, and CuSO₄. The ionsare believed to interact with the 2-O and 3-O sulfate groups in a mannernot yet fully understood to effectively shield them from desulfation.The degree of protection from desulfation is a function of ionconcentration, which is elaborated more in the discussion below.

Hydroxide ion is present in the reaction, preferably in the form of apaste of an alkaline earth or alkali metal salt, to increase the pH to alevel that initiates 2-O, 3-O desulfation of either heparin, or heparinfragments. One skilled in the art, using well known analyticaltechniques, including nuclear magnetic resonance (NMR) spectroscopy,will know to choose the preferred conditions, by monitoring the loss of2-O sulfate from iduronic acid. Similarly, the loss of 3-O sulfate fromGlcN can be monitored as a function of reaction conditions, as mentionedabove. The reaction mixture is dissolved in water and excess hydroxideion removed. The residue is purified to yield the product.

In some instances it might be beneficial to bleach the product to yielda white product.

More specifically, heparin or heparin fragments, and solid NaOH aremixed by grinding the solids together. Cold water is added to the cooledmixing vessel to form a homogenous paste. Depending on the degree of2-O, 3-O desulfation desired a known amount of copper is added. Areducing agent, preferably NaBH₄, may be added to prevent fragmentationand to maintain heparin in a substantially undegraded form, preferablyat pH's greater than 12.

0.2-0.8 g of hydroxide ion is added per gram of heparin. A small amountof cold water is added during the grinding process, and the grinding iscontinued in a vessel cooled at 0°-10° C. until a bright yellowhomogenous paste is obtained. The paste is allowed to sit at roomtemperature for a period of time (15 min-6 h, preferably 3 h). Theresidue is dissolved in water and excess hydroxide ion is removed,preferably using an ion-exchange resin (H+), or neutralization withacid. An aqueous solution of acetic or other mineral acids is preferred.The pH is adjusted to between 6-8. After exhaustive dialysis orultrafiltration, the solution is lyophilized to give substantiallyunfragmented 2-O, 3-O desulfated heparin (less than 10% of the startingmaterial is depolymerized), or 2-O, 3-O desulfated heparin fragments.Alternately, the product can be precipitated from solution by theaddition of ethanol using procedures well known in the art. Theoff-white product is optionally bleached to yield a final white solidproduct. Preferred bleaching conditions are those known in the art andare routinely used in heparin manufacturing process. The bleachingagents include, but are not limited to, potassium permanganate,peroxides and peracids.

The presence of bivalent metal ions in the reaction mixture causesdesulfation to occur inversely proportional to the concentration of ionspresent, and the duration of the reaction. The products from thisreaction are unique from other O-desulfated heparins in that onlyspecific sulfate groups are lost, altering the gross sulfate content andcharge of the product. It is important to note that this approach, andthe data generated by it could be used to produce a standard curve thatwould allow a skilled practitioner of this art to select a particularbivalent ion concentration that would yield a heparin composition havinga desired level of 2-O, 3-O desulfation. It will further be appreciatedthat such a standard curve could be produced by varying the heparinconcentration.

A key aspect of the instant invention, which partially distinguishes itfrom the work of Jaseja, M. et al. Can. J. Chem. (1989) 67: 1449-1456,is the reaction conditions, particularly the hydroxide ionconcentrations, that cause the loss of the 3-O-sulfate group from theglucosamine residues of heparin. It is important to be aware that since3-O-sulfated glucosamine is present in the ATIII binding sequence ofheparin, and is responsible for heparin's ATIII mediated anticoagulantactivity; consequently its loss yields a non-anticoagulant composition.

Another key aspect of the reaction conditions employed is the appearanceof free amine groups in the product (characterized by a resonance at 2.8ppm in the ¹ H-nmr spectrum at a pH >9).The free amine residues arisefrom hydrolysis of a portion of the 2-acetamide groups. Typically, 0-5%of total glucosamine appears as a free amine. The free amines can bereacted using known reactions in order to convert them to sulfaminogroups, acetamide groups, or other N-acyl groups. Thus, the instantinvention also encompasses N-modified heparin analogues.

N-modified heparins have been prepared from partially or completelyN-deacetylated heparin (Y. Guo and H. E. Conrad, Anal. Biochem., (1989)176:96-104; and Shaklee and Conrad Biochem J. (984) 217:187-197),followed by: (a) N-sulfation with appropriate N-sulfation reagents (L.Ayotte and A. S. Perlin, Carbohydr. Res. (1986) 145:267-277) to giveanalogues higher in sulfamino content and thus more anionic, (b)N-acylation with anhydrides (R(CO)₂ O, where R═--(CH₂)_(n) H and aryl)to yield analogues having hydrophobic substituents that may enhance inbinding to bioactive proteins by hydrophobic interaction. Additionalcompositions can also be produced by altering the sequence of thesereactions, such that the 2-O, 3-O desulfated heparin compositions of theinvention are subjected to N-deacetylation conditions, followed by re-Nsulfation or acylation by procedures known in the art.

Additional N-modified analogues can be prepared by partial or completeN-desulfation of heparin (L. Ayotte and A. S. Perlin, Carbohydr. Res.(1986) 145: 267-277) using known procedures, followed by re-N-acylationwith anhydrides (R(CO)₂ O, where R═--(CH₂)_(n) H and aryl) to yieldheparin compositions with reduced anionic charge. These compositions canalso be prepared from the 2-O, 3-O desulfated heparin compositions ofthe invention by N-desulfating the product and then re-N-acylating toyield the appropriate product analogue.

Labeled Forms of the Invention Non-Anticoagulant Compositions

The compositions of the invention can be provided with fluorescent,radioisotope, or enzyme labels as desired. Conventional techniques forcoupling of label to carbohydrates or related moieties can be used. Suchtechniques are well established in the art. See, for example, U.S. Pat.No. 4,613,665. The labeled mixtures of the invention may be used toidentify sites of disease as well as in competitive immunoassays, and asa means to trace the pharmacokinetics of the compositions in vivo.Suitable radioisotope labels for this purpose include hydrogen³,iodine¹³¹, indium¹¹¹, technetium⁹⁹, and phosphorus³². Suitable enzymiclabels include alkaline phosphatase, glucose-6-phosphate-dehydrogenase,and horseradish peroxidase. Particularly preferred fluorescent labelsinclude fluorescein and dansyl. A wide variety of labels of all threetypes is known in the art.

Administration and Use

The non-anticoagulant heparin compositions of the instant invention areuseful in therapeutic applications for treating or preventing a varietyof diseases including cancer, inflammation, and diseases caused orexacerbated by platelet aggregation, heparanase or angiogenic activity.The instant 2-O, 3-O desulfated heparin compositions, because of theiranti-angiogenic activity, will be preferably applied for the beneficialtreatment of angiogenic based diseases. One such class of diseases isretinopathies. A member of this class is diabetic retinopathy that willbe favorably treated by the compositions of the instant invention.

It should be noted that the preferred therapeutic composition consistsof 2-O, 3-O desulfated heparin fragments. Because of their reduced sizesuch fragments exhibit favored bioavailability and pharmacokineticproperties. See, Hirsh, J. and Levine, M., Blood (1992) 79:1-17.

Administration of either substantially unfragmented 2-O, 3-O desulfatedheparin, or 2-O, 3-O desulfated heparin fragments is typically by routesappropriate for glycosaminoglycan compositions, and generally includessystemic administration, such as by injection.

Particularly preferred is intravenous injection, as continuous injectionover long time periods can be easily continued. Also preferred areintroduction into the vascular system through intraluminaladministration or by adventitial administration using osmotic pumps orimplants. Typical implants contain biodegradable materials such ascollagen, polylactate, polylactate/polyglycoside mixtures, and the like.These may be formulated as patches or beads. Typical dosage ranges arein the range of 0.1-10 mg/kg/hr on a constant basis over a period of5-30, preferably 7-14, days. Particularly preferred dosage is about 0.3mg/kg/hr, or, for a 70 kg adult, 21 mg/hr or about 500 mg/day.

Other modes of administration are less preferred but may be moreconvenient. Injection subcutaneously at a lower dose or administeredorally at a slightly higher dose than intravenous injection, or bytransmembrane or transdermal or other topical administration forlocalized injury may also be effective. Localized administration througha continuous release device, such as a supporting matrix, perhapsincluded in a vascular graft material, is particularly useful where thelocation of the trauma is accessible.

Formulations suitable for the foregoing modes of administration areknown in the art, and a suitable compendium of formulations is found inRemington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., latest edition.

The compositions of the invention may also be labeled using typicalmethods such as radiolabeling, fluorescent labeling, chromophores orenzymes, and used to assay the amount of such compositions in abiological sample following its administration. Suitable protocols forcompetitive assays of analytes in biological samples are well known inthe art, and generally involve treatment of the sample, in admixturewith the labeled competitor, with a specific binding partner which isreactive with the analyte such as, typically, an immunoglobulin orfragment thereof. The antibodies prepared according to the invention, asdescribed below, are useful for this purpose. The binding of analyte andcompetitor to the antibody can be measured by removing the bound complexand assaying either the complex or the supernatant for the label. Theseparation can be made more facile by preliminary conjugation of thespecific binding partner to a solid support. Such techniques are wellknown in the art, and the protocols available for such competitiveassays are too numerous and too well known to be set forth in detailhere.

Antibodies may be prepared to 2-O, 3-O desulfated heparin, or 2-O, 3-Odesulfated heparin fragments by direct injection into an appropriateanimal host, or by coupling the compositions to suitable carders andadministering the coupled materials to mammalian or other vertebratesubjects in standard immunization protocols with proper inclusion ofadjuvants. Suitable immunogeuic carriers include, for example, KeyholeLimpet Hemocyanin (KLH), tetanus toxoid, various serum albumins such asbovine serum albumin (BSA) and certain viral proteins such as rotaviralVP6 protein. These coupled materials are then administered in repeatedinjections to subjects such as rabbits, rats or mice and antibody titersmonitored by standard immunoassays techniques. The resulting antiseramay be used per se or the antibody-secreting cells generated by theimmunization may be immortalized using standard techniques and used as asource of monoclonal preparations which are immunoreactive with 2-O, 3-Odesulfated heparin, or 2-O, 3-O desulfated heparin fragments.

Methods to conjugate 2-O, 3-O desulfated heparin, or 2-O, 3-O desulfatedheparin fragments to carders are known in the art. The compositions maybe linked to the carrier by, for example, homo- or heterobifunctionallinkers such as those marketed by Pierce Chemical Company, Rockford,Ill. Certain covalent linkers are described in U.S. Pat. No. 4,954,637.

Murine or human monoclonal preparations can be obtained by in vivo or invitro immortalization of peripheral blood lymphocytes or spleen cells ofanimals using methods well known in the art, such as fusion withimmortalizing cells as described by Kohler and Millstein Nature (1975)256:495; and Fendly, et al., Hybridoma (1987) 6:359. In vitro techniquesare generally described by Luben, R. and Mohler, M., MolecularImmunology (1980) 17:635; Reading, C. Methods in Enzymology (1986)121:18 (Part 1); or Voss, B., Methods in Enzymology (1986) 121:27.Recombinant and/or humanized antibody may also be generated usingmethods known in the art.

Properties of 2-O, 3-O Desulfated Heparin or 2-O, 3-O Desulfated HeparinFragments

As discussed above, heparin and non-anticoagulant heparins arebiologically active. Certain assays were conducted to determine thebiological properties of the instant invention compositions, and comparethese to the properties of heparin or non-anticoagulant heparins. Aparticularly noteworthy property of the 2-O, 3-O desulfated heparin isits low in vivo toxicity. The properties studied and the assays used aredescribed in detail in the Examples below.

The following examples are intended to illustrate but not to limit theinvention. For example, those skilled in the art would know that thereare materials and methods that can be substituted for those describedbelow, and still come within the scope of what is taught in theExamples.

EXAMPLE 1

Production of 2-O, 3-O Desulfated Heparin

1.0 g of Ming Han hog mucosal heparin was dissolved in 180 ml of waterand 20 ml of 1M NaOH was added to make the solution 0.1M in NaOH, and0.5% in heparin. The solution was frozen and lyophilized to dryness. Theresulting crusty yellow colored residue was dissolved in 50 ml of waterand then adjusted to pH 6-7 by the addition of 20% acetic acid solution.Solid sodium bicarbonate was added to bring the pH up to 8-9. Thesolution was exhaustively dialyzed and lyophilized thereby yielding 0.73g of solid product.

The product was subjected to various assays, including a determinationof APTT values, and disaccharide analysis. These assays were also usedin the following Examples, below. The values presented are relative tothe heparin starling material. A commercial kit obtained from Baxterlaboratories was used to determine APTT values. The manufacture'sinstructions regarding the use of the kit were followed. The values areexpressed as the % APTT relative to heparin.

The 2-O, 3-O desulfated heparin composition of the invention wascharacterized using ¹ H- and ¹³ C-nmr spectroscopy with detailedcompositional characterization performed by HPLC analysis of nitrousacid generated disaccharides, as described by Guo, Y. and Conrad, H. E.Analyt Biochem (1989) 176:96-104. Each sample was treated with 70%hydrazine and nitrous acid at pHs 1.5 and 4.0, and the resultingdisaccharides were quantified using the reversed phase ion pairing HPLCmethod described by Guo and Conrad, Anal. Biochem. (1988) 168, 54-62.

Using these assays the product isolated as described above exhibited thefollowing % anticoagulant activity (APTT), IdoA 2-S, and GMS₂,respectively; 10.0, 1.1, and <1.

EXAMPLE 2

Production of 2-O, 3-O Desulfated Heparin

Ming Han hog mucosal heparin (5 g) and solid NaOH (4 g) were mixed bygrinding the solids together in a vessel cooled at 0°-10° C. until abright yellow homogenous paste is obtained. A reducing agent (NaBH₄, 1g) was added to limit alkaline induced degradation of the heparinoid andthe product. A small amount of water (3 ml) was added during thegrinding process, and the mixing was continued until a bright yellowhomogenous paste was obtained. The paste was allowed to sit at roomtemperature for 3 h. Water was added to dissolve the paste, and thesolution was immediately neutralized to pH 7 by the addition of 20%acetic acid solution. The product was purified by ultrafiltration (MWCO1000 Da), and lyophilized to yield the final off-white product.

These compositions are essentially identical to the compositionsprepared by the lyophilization method described in Example 1, asdetermined by disaccharide analysis, MW!_(w), APTT and ¹ H-NMR.

EXAMPLE 3

Effect of Calcium on the Production of 2-O, 3-O Desulfated Heparin

1.0 g of Ming Han hog mucosal heparin was dissolved in 100 ml distilledwater, to which was added 250 mg of CaCl₂. This solution was mixed with100 ml of 0.2M sodium hydroxide solution, frozen and lyophilized todryness. The resulting crusty yellow colored residue was dissolved in 50ml of water and then adjusted to pH 6-7 by the addition of 20% aceticacid solution. Solid sodium bicarbonate was then added to bring the pHup to 8-9. The solution was then exhaustively dialyzed and afterlyophilization was isolated as a solid product (0.76 g). The %anticoagulant activity, IdoA 2-S, and GMS₂ were, respectively; 5.5, 3.3,and 1.9.

EXAMPLE 4

Effect of High Hydroxide Concentration on 2-O, 3-O Desulfation

Heparin (20.0 g, Ming Han hog mucosal was dissolved in 1000 ml of 0.4Msodium hydroxide solution, frozen and lyophilized to dryness. Theresulting crusty yellow colored residue was dissolved in 250 ml of waterand adjusted to pH 6-7 by the addition of 20% acetic acid solution.Solid sodium bicarbonate was added to bring the pH up to 8-9. Thesolution was then exhaustively dialyzed and after lyophilization wasisolated as a solid product (12.0 g). The % anticoagulant activity, IS,and GMS2 were, respectively; 8.0, <1.0, and <1.0.

EXAMPLE 5

Effect of Copper on the Production of 2-O, 3-O Desulfated Heparin

Experiments were conducted to show that the amount of 2-O, 3-Odesulfated heparin produced under the alkaline conditions of Example 1could be controlled by including copper in the reaction mixture. Thematerials and methods used in Example 3 were used here with theexception that copper II was substituted for 0.1M calcium, and the ratioof copper II to heparin disaccharide was varied.

FIG. 1 shows the per cent of residual ISMS and 3-O-sulfated GlcN (GMS2)remaining at the end of the reaction as a function of the copperII/heparin disaccharide mole ratio. It is apparent from FIG. 1 that theper cent of ISMS remaining correlates with the concentration of copperpresent in the reaction mixture. A similar relationship is seen forGMS2.

FIG. 2 shows the % APTT activity remaining at the concentrations ofcopper II tested: as the ratio of copper II to heparin disaccharideincreases the % APTT increases. Thus, it is concluded that copper IIprotects against 2-O, 3-O desulfation. It is important to note thatthese data could be used as a standard curve that would allow a skilledpractitioner of this art to select a particular copper II concentrationthat would yield a heparin composition having a desired level of 2-O,3-O desulfation.

EXAMPLE 6

Effect of Permanganate Bleaching on 2-O, 3-O Desulfated Heparin

Some deploymerization can occur when heparin is lyophilized fromalkaline solution to yield 2-O, 3-O-desulfated heparin or fractions andfragments thereof. The extent of depolymerization is related to theamount of excess base present during the lyophilization reaction, aswell as to the temperature reached during dissolution of the lyophilizedproduct and to the time in the alkaline solution prior toneutralization. It is believed that depolymerization occurs byβ-elimination reaction leading to a Δ-4,5-unsaturated uronic acidnon-reducing terminal residue. This is supported by the appearance of aresonance (5.8 ppm) in the ¹ H-NMR spectrum of the product that ischaracteristic of the Δ-4,5-unsaturated uronic acid residue formed byβ-elimination of heparin. The greater the extent of depolymerizationobserved, the greater the relative intensity of this resonance at 5.8ppm.

The 2-O, 3-O-desulfated heparin prepared according to the processdescribed in Example 1 was treated with 8% KMnO₄ solution at neutral pHfor 30-60 minutes at 50° C. The reaction was worked up under standardheparin manufacturing conditions to yield the bleached product. Thepermanganate bleaching of 2-O, 3-O-desulfated heparin results in amodification causing the loss of the Δ4,5 unsaturated residue. Thedisaccharide composition and other characterization data for the 2-O,3-O-desulfated heparin products are not substantially changed by thisbleaching step.

EXAMPLE 7

Production of 2-O, 3-O Desulfated Heparin Fragments

1.0 g of low molecular weight (5 kd) hog mucosal heparin, obtained fromCelsus Laboratories, was dissolved in 200 ml of 0.1M sodium hydroxidesolution. The solution was frozen and lyophilized to dryness, yielding acrusty yellow colored residue. The residue was dissolved in 50 ml ofwater and the solution adjusted to pH 6-7 by the addition of 20% aceticacid solution. Solid sodium bicarbonate was then added to bring the pHup to 8-9. The solution was exhaustively dialyzed and afterlyophilization was isolated as a solid product (0.77 g). The %anticoagulant activity was 1.2. IdoA 2-S and GMS₂ were not detectable,indicating that the heparin starting material is >99% desulfated.

EXAMPLE 8

Production of 2-O, 3-O Desulfated Heparin Fragments

5.0 g of low molecular weight (5 kd) hog mucosal heparin, obtained fromCelsus Laboratories and solid NaOH (4 g) were mixed by grinding thesolids together in a vessel cooled at 0°-10° C. until a bright yellowhomogenous paste is obtained. A reducing agent (NaBH₄, 1 g) was added tolimit alkaline induced degradation of the heparinoid and the product. Asmall amount of water (3 ml) was added during the grinding process, andthe grinding was continued until a bright yellow homogenous paste wasobtained. The paste was allowed to sit at room temperature for 3 h.Water was added to dissolve the paste, and the solution was immediatelyneutralized to pH 7 by the addition of 20% acetic acid solution. Theproduct was purified by ultrafiltration (MWCO 1000Da), and lyophilizedto yield the final off-white product.

These compositions are essentially identical to the compositionsprepared by the lyophilization method described in Example 7, asdetermined by disaccharide analysis, MW!_(w), APTT and 1H-NMR.

EXAMPLE 9

Effect of Limiting Hydroxide Ion Concentration on 2-O, 3-O Desulfationof Heparin Fragments

1.0 g of low molecular weight (5 Kd) hog mucosal heparin, obtained fromCelsus Laboratories, was dissolved in 200 ml of 0.05M sodium hydroxidesolution. This molarity is one half that used in the preceding example.The solution was frozen and lyophilized to dryness, yielding a crustyyellow colored residue. The residue was dissolved in 50 ml of water andthe solution adjusted to pH 6-7 by the addition of 20% acetic acidsolution. Solid sodium bicarbonate was then added to bring the pH up to8-9. The solution was exhaustively dialyzed and after lyophilization wasisolated as a solid product (0.77 g). The % anticoagulant activity, IdoA2-S, and GMS2 were, respectively; 23, 12.1, and 3.8.

EXAMPLE 10

Production of 2-O, 3-O Desulfated Heparin From Re-N-SulfatedN-Deacetylated Heparin

1.0 g of re-N-sulfated N-deacetylated heparin produced as described byLloyd, A. G. et. al., Biochem. Pharmacol (1971) 20:637-648 and Guo, Y.and Conrad, H. E., Anal Biochem (1989) 176:96-104 was dissolved in 200ml of 0.1M sodium hydroxide solution, frozen and lyophilized to dryness.The resulting crusty yellow colored residue was dissolved in 50 ml ofwater and adjusted to pH 6-7 by the addition of a 20% acetic acidsolution. Solid sodium bicarbonate was added to bring the pH up to 8-9,and the solution was exhaustively dialyzed and lyophilized, yielding0.73 g of a solid product.

The % anticoagulant activity, IdoA 2-S, and GMS2 were, respectively;9.5, 1.2, and <1.0.

EXAMPLE 11

Anti-Angiogenic Activity

Compounds that stimulate or inhibit angiogenesis can be identified usingseveral assays known in the art. The heparinoids of the instantinvention were tested using the chicken chorioallantoic membrane (CAM)assay. The assay was performed as described by Castellot et. al., J. ofCellular Physiology (1986) 127:323-329, with the exception that sampleswere evaluated for their efficacy to inhibit neovascularization. Agarosepellets containing 50 μg of hydrocortisone, or hydrocortisone plusdifferent amounts of 2-O, 3-O desulfated heparin were incubated on theCAM for 3-4 days before scoring the results. 2-O, 3-O desulfated heparinwas produced as described in Example 1.

Table 1 shows the results. It is apparent that the 2-O, 3-O desulfatedheparin composition exhibits angiostatic activity. Angiostatic activityis defined as a partial clearing or an avascular zone around the pellet.In all cases, pellets at each heparinoid concentration contained 50 μgof hydrocortisone.

The number in parenthesis in the Table is the percent of the totalembryos scored that exhibited no effect, a partial clearing, or anavascular zone. For example, 3.125 μg/ml of the 2-O, 3-O desulfatedheparin had no effect on 20 embryos and a partial clearing on 4 embryos.Thus, under these conditions 83.3% of the embryos showed no effect and16.7% exhibited a partial clearing.

                  TABLE 1                                                         ______________________________________                                        Chick Chorioallantoic Membrane Bioassay                                       Compound 2-O, 3-O Desulfated Heparin                                          Composition (μg/ml)             Avascular                                  Non-anticoagulant                                                                          No Effect Partial Clearing                                                                          Zone                                       heparin      (0)       (+)         (++)                                       ______________________________________                                        0.00         22    (100.0)                                                    3.125        20    (83.3)  4    (16.7)                                        6.25         16    (59.3)  9    (33.3) 2   (7.4)                              12.5         9     (36.0)  10   (40.0) 6   (24.0)                             25.0         14    (58.3)  10   (41.7)                                        50.0         12    (41.4)  13   (44.8) 4   (13.8)                             ______________________________________                                    

EXAMPLE 12

Heparanase Inhibitory Activities of 2-O, 3-O and 2-O Desulfated HeparinCompositions

The 2-O, 3-O desulfated heparin composition of the instant invention wastested for heparanase inhibitory activity using heparanase from a rathepatoma cell line. The cell line is described by Gerschenson, et al.,Science (1970) 170:859-861. Further, its inhibitory activity wascompared to 2-O desulfated heparin of Jaseja, M. et al., Can. J. Chem.(1989) 67:1449-1456. Recall that this composition is produced from beeflung heparin, unlike the instant compositions which are produced fromhog mucosal heparin. 2-O, 3-O desulfated heparin was produced asdescribed in Example 1, and the 2-O desulfated heparin composition wassupplied by Dr. Perlin, a co-author of the Jaseja, M. et al publication,above.

The procedures for isolating heparanase from hepatoma cells, and themethods for assaying the activity of the enzyme are known by thoseskilled in the art. The following procedures and materials were used.

Confluent rat hepatoma cell cultures were grown in standard cell cultureflash, and washed 3 times with 10 ml of a 50 mM Hepes solutioncontaining 0.25M sucrose and 0.14 M NaCl, pH 7.4. Next, 1 ml of a 50 mMMES buffer pH 5.2, containing 0.14M NaCl, 6 mM sodium azide, and certainprotease inhibitors was added and the cells removed from the flask usinga disposable cell scraper. The following protease inhibitors werepresent in the MES buffer: 0.2 μg/ml aprotinin, 0.5 μg/ml leupeptin, 100μg/ml soybean trypsin inhibitor, 1 mM PMSF, 2 mM EDTA (sodium salt), and15 mM D-saccharic acid 1,4 lactone (exoglucuronidase inhibitor).

The cells were added to a 7 ml Dounce homogenizer, freezed/thawed 3times in an ethanol/dry ice bath, and homogenized with 15 strokes usinga tight pestle. The resulting cell lysates were placed in a 2 mlcentrifuge tube and centrifuged at 4° C. for 30 minutes at 16,000×g. Thesupernatant was removed, and the protein concentration in thesupernatant determined using the Macro BCA protein assay. BSA was usedas a standard.

Heparanase activity was quantified by measuring soluble N-³ H-acetylatedpancreas heparan sulfate fragments derived from uncleaved N-³H-acetylated pancreas heparan sulfate by cetylpyridinium chloride (CPC)precipitation. N-³ H-acetylated pancreas heparan sulfate had a weightaverage molecular weight, or Mw, of about 12,000. The followingprocedures were used.

Rat hepatoma cell supernatant, isolated as described above, containing10 μg of protein was brought up to 30 μl with 50 mM MES buffer pH 5.2containing 0.14M NaCl, 6 mM sodium azide and the protease inhibitorsdescribed above, and added to siliconized 1.5 ml microcentrifuge tubes.Next, ³ H-acetylated pancreas heparan sulfate (93 ng, 30,000 cpm) in 10μl of 200 mM MES buffer pH 5.2 containing 0.14M NaCl was added to tubescontaining the rat hepatoma cell supernatant. 10 μl of distilled watercontaining various concentrations of 2-O, 3-O desulfated heparin, or 2-Odesulfated heparin of Jaseja, M., et al., above, was added. The assaywas run in triplicate for each inhibitor concentration. Three "0" timepoints were run as controls in which no inhibitor was added. It waspreviously shown that the highest concentration of inhibitor does notaffect precipitation of the intact radiolabeled heparan sulfatesubstrate.

The enzyme substrate inhibitor mixture was spun in a microcentrifuge,after which the tubes were incubated at 37° C. for 30 minutes. The "0"time points were maintained on ice. After the appropriate time, thereaction was stopped by adding to the reaction tubes the following:

1) 150 μl of an aqueous heparin solution (0.33 mg/ml)

2) 200 μl of 100 mM sodium acetate pH 5.5

3) 100 μl of CPC (0.6% in water)

Next, the tubes were vortexed, incubated for 60 minutes at roomtemperature, and then centrifuged for 10 minutes at 4,000×g in a 5415CEppendoff centrifuge. The supernatant was removed and assayed for ³ H byliquid scintillation counting.

FIG. 3 shows the results. The 2-O, 3-O desulfated heparin composition ofthe instant invention is denoted GM 1603 while the 2-O desulfatedcomposition of Jaseja, M., et al., is denoted GM1869. It is apparentthat the 2-O, 3-O desulfated heparin composition is significantly moreactive than the 2-O desulfated composition. Indeed, the IC₅₀ values were4.0 μg/ml and 7.6 μg/ml, respectively.

Thus, these results establish that the 2-O, 3-O desulfated heparincomposition of the instant invention is a heparanase inhibitor, andfurther, it is significantly more active than the 2-O desulfated heparincomposition of Jaseja, M. et al.

EXAMPLE 13

Activity of 2-O, 3-O Desulfated Heparin in bFGF Binding Assay

A cell based assay as described by Ishihara, M. et al., AnalyticalBiochemistry (1992) 202:310-315 was used to measure the effect of 2-O,3-O desulfated heparin on bFGF binding. 2-O, 3-O desulfated heparin wasproduced as described in Example 1. Similar experiments were run usingMing Han hog mucosal heparin and the non-anticoagulant heparincomposition produced as described in U.S. Pat. No. 5,280,016, or PCTPatent Application No. US92/02516, filed Mar. 27, 1992. This compositionconsists of heparin oxidized with sodium periodate and subsequentreduction with sodium borohydride.

The assay is based on the observation that bFGF binds to alymphoblastoid cell line, RO-12, that expresses hamster syndecan, andthat this interaction can be inhibited by compounds that bind to bFGF.The cell line is transfected with cDNA that encodes the core protein ofsyndecan. Hamster syndecan is known to bind bFGF, presumably becauseheparan sulfate chains are bound to the core protein by the RO-12 cellline.

The assay was run as follows. Fifty microliters of 10 μg/ml humanrecombinant bFGF was added to wells of a 96-well tissue culture plateand incubated overnight at 4° C. The wells were aspirated with PBS toremove any unbound bFGF, rinsed twice with PBS, and subsequentlyincubated with PBS containing 5% (v/v) fetal bovine serum for 1 hour atroom temperature. RO-12 cells were suspended at a density of 3×10⁶cells/ml in PBS containing 5% fetal bovine serum. To this mixture wasadded the desired amount of 2-O, 3-O desulfated heparin, or heparin. Thecompositions were used, in μg/ml, at concentrations of 50, 25, 12.5,6.3, 3.1, 1.6, and 0.8. They were made up in PBS plus 2.5% fetal bovineserum. A control was also run, containing only PBS plus 2.5% fetalbovine serum. Next, 100 μl of the cell mixture was immediately added tothe microtiter wells, and incubated for 5 minutes, after which the wellswere washed 3 times with PBS. Finally, the amount of cell protein boundto the wells was determined by dissolving the cell pellets in 20 μl of5% SDS and measuring the protein concentration of the cell lysates. BSAwas used as the standard.

The results established that the concentrations of 2-O, 3-O desulfatedheparin, the non-anticoagulant heparin composition produced as describedin U.S. Pat. No. 5,280,016, or heparin that inhibits 50% of cell bindingto bFGF were >50 μg/ml, <1 μg/ml and <1 μg/ml, respectively. Thus, 2-O,3-O desulfated heparin composition of the instant invention has greatlyreduced binding activity to bFGF relative to heparin, or thenon-anticoagulant heparin composition described in U.S. Pat. No.5,280,016.

EXAMPLE 14

Effect of 2-O, 3-O Desulfated Heparin on Ristocetin Induced PlateletAggregation

The effect of 2-O, 3-O desulfated heparin on ristocetin induced plateletaggregation was measured in the presence of vWF as described by Sobel etal., J. Clin. Invest. (1992) 87:1787-1793, and Kelton et al., Thromb Res(1980) 18:477-483. Also tested were the effects of Ming Han hog mucosalheparin, and the NAC heparin described in U.S. Pat. No. 5,280,016, orPCT Patent Application No. US92/02516, filed Mar. 27, 1992. 2-O, 3-Odesulfated heparin was produced as described in Example 1.

The experiment was conducted as follows. Platelet-rich plasma wasprepared from citrated whole blood of 300-500 gram male guinea-pigs bylow speed centrifugation to sediment the red blood cells. The guineapigs were anesthetized with methoxyflurane. The upper layer washarvested and used to determine the effects of the heparinoids onplatelet aggregation. The remaining red blood cell rich plasma wascentrifuged at high speed in order to prepare a platelet poor plasmafraction that was used as a blank in the aggregometer.

400 μl samples, consisting of 200 μl of platelet-rich plasma and 200 μlof platelet poor plasma, were placed in the light path of a dualaggregation module (Payton) two-channel aggregometer, and preincubatedat 37° C. with various concentrations of heparinoid test material or PBSbuffer control for 10 minutes. The samples were continuously stirred at1,000 rpm. Aggregation was induced by adding 6 μl of ristocetin (stocksolution, 125 mg/ml in 0.9% sterile saline) and aggregation recorded asthe change in light transmission using the platelet poor plasma as ablank.

2-O, 3-O desulfated heparin and heparin were tested at variousconcentrations, the highest being 1000 μg/ml, and the remaining being 2fold serial dilutions.

The results were expressed as the EC₇₀ concentrations, or theconcentration that was effective at inhibiting 70% aggregation. The EC₇₀concentrations for 2-O, 3-O desulfated heparin and for the NAC heparindescribed in U.S. Pat. No. 5,280,016, were expressed relative to heparinwhich was taken as 1. The EC₇₀ concentrations for 2-O, 3-O desulfatedheparin and for the NAC heparin shown in the patent application were 0.4and 2-4, respectively.

Thus, the 2-O, 3-O desulfated heparin composition of the instantinvention inhibits platelet aggregation to a lesser extent than heparinindicating possibly reduced bleeding potential.

EXAMPLE 15

Toxicity of 2-O, 3-O Desulfated Heparin Compositions

Experiments were done to determine the in vivo toxicity of the 2-O, 3-Odesulfated heparin compositions. A group of three (3) mice wasadministered 2-O, 3-O desulfated heparin subcutaneously, once a dayaccording to the following schedule: 20 mg/kg on day 1, 40 mg/kg on day2, 80 mg/kg on day 3, and 160 mg/kg on day 4. At the end of day 4, two(2) of the mice showed no signs of toxicity while the third, althoughhealthy, presented subcutaneous swelling at the site of injection. Basedon these results, and considering the high doses used, there is littleor no in vivo toxicity associated with the 2-O, 3-O desulfated heparincompositions.

EXAMPLE 16

Effect of 2-O, 3-O Desulfated Heparin Compositions on Tumor Growth

Experiments were conducted to test the efficacy of 2-O, 3-O desulfatedheparin compositions on tumor growth in an animal model system, the nudemouse, that closely mimics the human condition. Two human tumor celllines were utilized; a pancreatic adenocarcinoma, CAPAN-2, and a mammaryadenocarcinoma, MDA231. Both cell lines grow aggressively in nude mice,and CAPAN-2 exhibits the multiple drug resistant phenotype.

The experiments were conducted as follows: male, 20 gram, nude mice, ingroups of ten, were inoculated subcutaneously with 3×10⁶ viable CAPAN-2cells in 0.2 ml PBS/matrigel (1:3). Twenty four hours latter, the micewere subcutaneously injected with 2-O, 3-O desulfated heparin at a doseof 60 mg/kg, made up in PBS, and produced as described in Example 1.Control mice were injected with PBS vehicle only. Experimental andcontrol mice were injected daily for 36 days, after which tumor volumewas determined using standard methods.

The results are shown in FIG. 4. It is apparent that there issignificant anti-tumor activity of the 2-O, 3-O desulfated heparincomposition, denoted NAC 6 in the figure, starting at about day 20, andcontinuing to day 31 post tumor challenge.

Similar experiments were conducted in nude mice using the mammaryadeno-carcinoma, MDA231, cell line. Mice were dosed at 50 mg/kg/day. Theresults revealed that 2-O, 3-O desulfated heparin compositionsignificantly inhibited the growth of the tumor cells.

EXAMPLE 17

Effect of Sodium Hydroxide Concentration on the Production of 2-O, 3-ODesulfated Heparin Fragments

Experiments were conducted using heparin fragments to show that theextent of 2-O, 3-O desulfation of such fragments is a function of theconcentration of sodium hydroxide used to carry out the reaction. Inprevious experiments this was shown for substantially undepolymerizedheparin.

The materials and methods used were essentially those set forth in therelevant preceding examples. Note though that 2.0 g of low molecularweight (3 Kd) hog mucosal heparin was used for the experiments. Table 2lists certain of the reaction conditions including the concentrations ofsodium hydroxide, yield of the reaction products, % APTT, anddisaccharide analysis. The latter two parameters are plotted in FIG. 5as function of sodium hydroxide concentration.

From FIG. 5 it is apparent that APTT activity of the reacted fragmentsrelative to control low molecular weight heparin gradually declines overthe concentrations of sodium hydroxide tested, and approaches zero atabout 1M sodium hydroxide. Measurements of the level of 2-O, 3-Osulfation of the fragments revealed a greater loss of 2-O sulfateresidues at lower sodium hydroxide concentrations than that observed for3-O sulfate residues with nearly total 2-O desulfation at sodiumhydroxide concentrations of 0.5M or greater. Over the same concentrationof sodium hydroxide, more 3-O sulfate residues were maintained than 2-Osulfate residues, but as shown in the figure at 1M sodium hydroxide only30-35% of the original 3-O sulfate resides present in the 3 Kd fragmentsare maintained.

Having described what the applicants believe their invention to be, askilled practitioner of this art should not construe the invention to belimited other that by the scope of the appended claims.

We claim:
 1. A composition comprising unfragmented 2-O, 3-O desulfatedheparin, or 2-O, 3-O desulfated heparin fragments wherein saidunfragmented 2-O, 3-O desulfated heparin, or 2-O, 3-O desulfated heparinfragments are from 80% to 99% and from 24% to 75% desulfated at the 2-Oand 3-O positions, respectively, and wherein said 2-O, 3-O desulfatedheparin fragments have an average molecular weight range of 2-6.5 kd. 2.The composition of claim 1 wherein said composition comprises said 2-O,3-O desulfated heparing fragments.
 3. The composition of claim 2 whereinsaid fragments have an average molecular weight range of about 5 kd. 4.A pharmaceutical composition comprising unfragmented 2-O, 3-O desulfatedheparin, or 2-O, 3-O desulfated heparin fragments wherein saidunfragmented 2-O, 3-O desulfated heparin, or 2-O, 3-O desulfated heparinfragments are from 80%. to 99% and from 24% to 75% desulfated at the 2-Oand 3-O positions, respectively, and wherein said 2-O, 3-O desulfatedheparin fragments have an average molecular weight range Of 2-6.5 kd. 5.The pharmaceutical composition of claim 4 wherein said compositioncomprises said 2-O, 3-O desulfated heparing fragments.
 6. Thepharmaceutical composition of claim 4 wherein said unfragmented 2-O, 3-Odesulfated heparin or 2-O, 3-O desulfated heparin fragments have artaverage molecular weight range of about 5 kd.
 7. The composition ofclaim 1, wherein said 2-O, 3-O desulfated heparin fragments are about65% desulfated at the 3-O position.
 8. The composition of claim 7wherein said composition has APTT activity of about 10% of heparin.
 9. Acomposition comprising unfragmented 2-O, 3-O desulfated heparin or 2-O,3-O desulfated heparin fragments that are from 80% to 99% and from 24 %to 75 % desulfated at the 2-O and 3-O positions, respectively, andwherein said 2-O, 3-O desulfated heparin fragments have an averagemolecular weight range Of 2-6.5 kd, prepared by:a) admixing in solutionheparin or heparin fragments and an amount of bivalent metal cation thatis sufficient to produce the desired amount of desulfation, saidsolution having an alkaline pH; and b) freezing and lyophilizing saidsolution.
 10. The composition of claim 9 wherein said alkaline pH isfrom 11-14.
 11. The composition of claim 10 wherein said pH is
 13. 12.The composition of claim 11, wherein a reducing agent is added to saidsolution in step a).